There is an urgent need for new drugs to treat HIV/AIDS. Of particular importance is the discovery and development of compounds that are active against virus isolates resistant to currently approved therapies. We recently reported on 3-O-(3',3'-dimethylsuccinyl) betulinic acid (PA-457), first in a new class of HIV-1 inhibitors that block virus replication by disrupting virus maturation. Unlike protease inhibitors, PA-457 blocks a single step in the processing of the viral Gag protein. Specifically, PA-457 disrupts the cleavage of the Gag capsid (CA) precursor (CA-SP1) to mature CA protein resulting in the release of immature, non-infectious viral particles. Importantly, PA-457's distinct mechanism of action allows the compound to retain potent activity against HIV-1 strains resistant to the current classes of approved drugs. Work by our group and others suggest that residues within the HIV-1 Gag CA-SP1 domain serve as determinants of PA-457 activity and genetic variation within this domain allows HIV-1 to escape PA-457- mediated inhibition. Most recent results support the theory that a direct interaction between the compound and an oligomeric form of Gag is critical to PA-457 activity. While these observations allow some insights into the PA-457 antiviral effect, the mechanisms of action and resistance to PA-457 remain to be fully determined. To address these issues, we propose the following specific aims: (1) To further characterize the mechanism of action of PA-457 activity, and (2) To further characterize the molecular determinants of PA-457 activity. We believe that the results of these studies will provide insights into the mechanisms of action and resistance of PA- 457. A better understanding of both of these issues is particularly relevant due to PA-457's ongoing clinical development. We are also confident that the results of these studies will be directly applicable to the design of additional broadly active HIV-1 maturation inhibitors effective against both WT HIV-1 strains and the resistant virus isolates that emerge in PA-457-treated patients. Drug resistance is the leading reason for HIV treatment failure. Successful completion of the proposed research will help identify and design additional HIV maturation inhibitors that will provide additional treatment options and improve disease outcome. [unreadable] [unreadable] [unreadable] [unreadable]